| 摘要: | 人類多能幹細胞 (hPSCs) 可以分為人類胚胎幹細胞 (hESCs) 和人類誘導多能幹細胞 (hiPSCs)，這些細胞可以分化成源自三個胚層的細胞，如內胚層、中胚層和外胚層。一般而言，與大多數癌細胞或組織細胞不同，hPSCs 無法在傳統的組織培養聚苯乙烯 (TCPS) 盤上增殖。而 hPSCs 通常在塗有 Matrigel 的培養皿上培養，其中包含異種物質。因此有必要使用嵌合合成肽的生物材料來促進 hPSC 的粘附、多能性的保持和分化，以支持hPSC 的臨床應用。為了維持 hPSCs 的增殖，我們開發了具有最佳彈性 (25.3 千帕) 的特定肽嵌合的 PVA-IA（聚（乙烯醇-乙烯酸酯-巯基丙烯酸））水凝膠，其中以特定的層黏連蛋白-β4 和玻連蛋白衍生的寡肽嵌合的水凝膠是最適合 hPSC 增殖的生物材料。在我們之前的研究中，hPSCs 可以在嵌合了特定層黏連蛋白-β4 和玻連蛋白衍生寡肽的水凝膠上成功培養超過 10 代。然而用於嵌合的寡肽溶液濃度 (通常為 1000 μg/mL) 比 ECM 蛋白質塗層表面溶液的濃度 (通常為 5-10 μg/mL) 要高得多。為了改進這一點，本研究開發了一種新的基於樹枝狀結構的肽嵌合表面細胞培養生物材料設計。由多個支鏈的醯胺和胺基組成的第 3 代聚胺酯胺（PAMAM）樹枝狀結構被固定在水凝膠表面，預期對 hPSC 的培養和分化具有高生物相容性。將嵌合不同肽濃度的 CLB1GK (CGGGGKGGPMQKMRGDVFSP) 的樹枝狀結構表面上的 hiPSCs 細胞增殖進行定量評估。探究哪種肽濃度更適合hiPSCs 的增殖。hPSCs 可以在低濃度的肽 (50 μg/ml) 嵌合的樹枝狀結構表面增殖。在肽濃度較低（50 μg/ml）的條件下，在肽嵌合的樹枝狀結構表面長期培養（10 代）的 hiPSCs 表現出其多能性和分化為三個胚層細胞的能力。目前，只有這項研究支持 hPSC 在肽濃度較低的肽嵌合樹枝狀結構表面上增殖，而大多數用於 hPSC 培養的典型肽嵌合生物材料是使用肽濃度較高的溶液，例如>1000 μg/ml。在樹枝狀結構表面3D 嵌合多肽能夠在長時間的培養中維持 hPSC 的多能性。;Human pluripotent stem cells (hPSCs) can be divided into human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), which can differentiate into the cells derived from three germ layers, such as endoderm, ectoderm, and mesoderm. Typically, conventional tissue culture polystyrene plates cannot support the proliferation of hPSCs, unlike most cancer cells or tissue cells that can proliferate. Alternatively, hPSCs are commonly cultured on dishes coated with Matrigel, which involves xeno-containing substances. Hence, it is advisable to employ biomaterials grafted with synthetic peptides to facilitate hPSC adhesion, maintain hPSC pluripotency, and differentiate hPSCs for the clinical application of hPSCs. The specific peptide-grafted PVA-IA (poly (vinyl alcohol-co-vinyl acetate-co-itaconic acid)) hydrogels with optimal elasticity (25.3 kPa) have been developed to support the proliferation of hPSCs, where the hydrogels grafted with specific oligopeptides derived from laminin-β4 (LMN) and vitronectin were the most preferable biomaterials for hPSC proliferation and could be used as cell culture biomaterials of hPSCs for more than 10 passages. However, the concentration of oligopeptide solution needed for grafting on the hydrogels should be much higher (typically 1000 μg/mL) than the concentration of ECM proteins used for the preparation of ECM protein-coated surface (typically 5-10 μg/mL). To improve this point, a new design of cell culture biomaterials using a dendrimer-based peptide-grafted surface was developed in this study. Polyamidoamine (PAMAM) dendrimer having generation 3, which was composed of many branched subunits of amide and amine groups, was used to be immobilized on the hydrogel surface, where it is expected to have high biocompatibility for hPSC culture and differentiation. The cell proliferation of hiPSCs on PAMAM dendrimer surface grafted with CLB1GK with different peptide concentration were evaluated from expansion fold. It is evaluated which peptide concentration was more suitable for hiPSC proliferation in this study. The hPSCs could be cultured on PAMAM dendrimer surface grafted with the peptide, which were prepared with low concentration of peptide (50 μg/mL) and where the concentration of peptide is the same order of the concentration of coating ECM solution (5-10 μg/mL) on ECM protein-coated surface. The hiPSCs showed their pluripotency and differentiation ability into three germ layer cells after long-term (10 passage) culture on the peptide-grafted PAMAM dendrimer hydrogel surface, which were prepared with a low peptide concentration (50 μg/ml). Currently, only this study supports hPSC proliferation on the peptide-grafted surface, which was prepared with a low concentration of peptide solution (50 μg/ml), whereas most typical peptide-grafted biomaterials for hPSC culture was prepared with a high concentration of peptide solution, such as >1000 μg/ml. 3D peptide location (immobilization) on the surface using PAMAM dendrimer enables to maintain the pluripotency of hPSCs during their cultivation for
a long time. |
